ldn-193189 and Disease-Models--Animal

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant

Topics: Animals; Blast Injuries; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Burns; Cell Differentiation; Disease Models, Animal; Humans; Immunoconjugates; Immunoglobulin Fc Fragments; Ligands; Lower Extremity; Male; Mesenchymal Stem Cells; Mice, Inbred C57BL; Ossification, Heterotopic; Osteogenesis; Pyrazoles; Pyrimidines; Rats, Sprague-Dawley; Signal Transduction; Wounds and Injuries; X-Ray Microtomography

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext. We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice.. Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007).. HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Topics: ADAMTS5 Protein; Animals; Bone Morphogenetic Proteins; Cartilage, Articular; Chondrocytes; Disease Models, Animal; Heparitin Sulfate; Hypertrophy; Matrix Metalloproteinase 13; Mice; Mice, Knockout; Mice, Transgenic; N-Acetylglucosaminyltransferases; Osteoarthritis, Knee; Pyrazoles; Pyrimidines; Smad1 Protein; Smad5 Protein; Smad8 Protein

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Density; Bone Diseases; Bone Marrow; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Cell Line, Tumor; Disease Models, Animal; Femur; Gene Expression Profiling; Gene Expression Regulation; Humans; Injections, Intraperitoneal; Mice; Mice, Inbred Strains; Multiple Myeloma; Osteoclasts; Osteogenesis; Pyrazoles; Pyrimidines; RNA-Seq; Signal Transduction; Stem Cells; Tibia; Treatment Outcome; Xenograft Model Antitumor Assays

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Topics: Animals; Bone Morphogenetic Proteins; Cervical Cord; Chondrogenesis; Disease Models, Animal; Embryonic Development; Exostoses, Multiple Hereditary; Growth Plate; Heparitin Sulfate; Humans; Magnetic Resonance Imaging; Mice; Mice, Knockout; Mutation; N-Acetylglucosaminyltransferases; Osteochondroma; Pyrazoles; Pyrimidines; Smad1 Protein; Tomography, Emission-Computed

Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Angiotensin II; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrosis; Inhibitor of Differentiation Protein 1; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; NFATC Transcription Factors; Phenylephrine; Phosphorylation; Pyrazoles; Pyrimidines; Rats, Sprague-Dawley; RNA Interference; Signal Transduction; Smad Proteins; Time Factors; Transfection

Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ≈2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice.. We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification.. BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

Topics: Animals; Aortic Valve; Aortic Valve Stenosis; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Calcinosis; Cells, Cultured; Chondrogenesis; Disease Models, Animal; Extracellular Matrix; Female; Fibroblast Growth Factor-23; Gene Expression Regulation; Genetic Predisposition to Disease; Glucuronidase; Klotho Proteins; Male; Mice, Knockout; Osteogenesis; Phenotype; Phosphorylation; Pyrazoles; Pyrimidines; Signal Transduction; Smad1 Protein; Smad5 Protein; Smad8 Protein; Swine

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenetic disease that still lacks effective therapy. Repulsive guidance molecule b (RGMb), a co-receptor for bone morphogenetic proteins (BMPs) and a ligand for neogenin, is expressed in renal tubular epithelial cells. Previous studies showed that RGMb plays negative roles in several types of tumors and prevents the immune system from over activation. The present study was designed to explore the effects of RGMb in ADPKD development. We found that expression of RGMb in kidney was less in PKD mice than wild-type mice. With stimulation of 8-bromo-cAMP, RGMb-null embryonic kidneys had greater cyst index, though their ureteric bud branched less than wild-type mice at E13.5. Postnatal RGMb-null kidneys showed interstitial hyperplasia and decreased tubular structures, especially in the boundary area of renal cortex and medulla. RGMb overexpression dramatically inhibited cyst development and promoted tubulogenesis in MDCK cells grown in 3D collagen gels. Biochemical analysis showed increased p-Smad1/5/8 and decreased p-ERK in RGMb-overexpressing MDCK cells, suggesting modulated BMP signaling. Specific inhibition of p-Smad1/5/8 by LDN193189 reversed the suppression of RGMb on MDCK cyst model. These results reveal RGMb as a novel regulator for ADPKD by promoting renal tubule branching and regulating BMP signaling pathway. Elevating RGMb and enhancing p-Smad1/5/8 are promising new strategies to treat ADPKD.

Topics: Animals; Apoptosis; Bone Morphogenetic Proteins; Cell Adhesion Molecules, Neuronal; Cell Proliferation; Cytoprotection; Disease Models, Animal; Dogs; Extracellular Signal-Regulated MAP Kinases; GPI-Linked Proteins; Kidney Diseases, Cystic; Kidney Tubules; Madin Darby Canine Kidney Cells; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Phosphorylation; Pyrazoles; Pyrimidines; Signal Transduction; Smad Proteins; TRPP Cation Channels

Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients.. We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model.. Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes.. Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD.

Topics: Adenine; Anemia, Iron-Deficiency; Animals; Anti-Infective Agents; Blotting, Western; Bone Morphogenetic Proteins; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Erythropoiesis; Hepcidins; Humans; Iron; Kidney Diseases; Male; Pyrazoles; Pyrimidines; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis.. To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E-/- mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O-positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects.. These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

Topics: Animals; Antimicrobial Cationic Peptides; Apolipoproteins E; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; Bone Morphogenetic Proteins; Cell Differentiation; Cholesterol; Disease Models, Animal; Foam Cells; Hepcidins; Iron; Lipoproteins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyrazoles; Pyrimidines; Signal Transduction

The expression of bone morphogenetic proteins (BMPs) is enhanced in human atherosclerotic and calcific vascular lesions. Although genetic gain- and loss-of-function experiments in mice have supported a causal role of BMP signaling in atherosclerosis and vascular calcification, it remains uncertain whether BMP signaling might be targeted pharmacologically to ameliorate both of these processes.. We tested the impact of pharmacological BMP inhibition on atherosclerosis and calcification in LDL receptor-deficient (LDLR-/-) mice. LDLR-/- mice fed a high-fat diet developed abundant vascular calcification within 20 weeks. Prolonged treatment of LDLR-/- mice with the small molecule BMP inhibitor LDN-193189 was well-tolerated and potently inhibited development of atheroma, as well as associated vascular inflammation, osteogenic activity, and calcification. Administration of recombinant BMP antagonist ALK3-Fc replicated the antiatherosclerotic and anti-inflammatory effects of LDN-193189. Treatment of human aortic endothelial cells with LDN-193189 or ALK3-Fc abrogated the production of reactive oxygen species induced by oxidized LDL, a known early event in atherogenesis. Unexpectedly, treatment of mice with LDN-193189 lowered LDL serum cholesterol by 35% and markedly decreased hepatosteatosis without inhibiting HMG-CoA reductase activity. Treatment with BMP2 increased, whereas LDN-193189 or ALK3-Fc inhibited apolipoprotein B100 secretion in HepG2 cells, suggesting that BMP signaling contributes to the regulation of cholesterol biosynthesis.. These results definitively implicate BMP signaling in atherosclerosis and calcification, while uncovering a previously unidentified role for BMP signaling in LDL cholesterol metabolism. BMP inhibition may be helpful in the treatment of atherosclerosis and associated vascular calcification.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Atherosclerosis; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Cardiovascular Agents; Cholesterol, LDL; Diet, High-Fat; Disease Models, Animal; Endothelial Cells; Fatty Liver; Female; Hep G2 Cells; Humans; Lipoproteins, LDL; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyrazoles; Pyrimidines; Reactive Oxygen Species; Receptors, LDL; Recombinant Fusion Proteins; Signal Transduction; Time Factors; Vascular Calcification

Anemia of chronic inflammation (ACI) is the most frequent anemia in hospitalized patients and is associated with significant morbidity. A major underlying mechanism of ACI is the retention of iron within cells of the reticuloendothelial system (RES), thus making the metal unavailable for efficient erythropoiesis. This reticuloendothelial iron sequestration is primarily mediated by excess levels of the iron regulatory peptide hepcidin down-regulating the functional expression of the only known cellular iron export protein ferroportin resulting in blockade of iron egress from these cells. Using a well-established rat model of ACI, we herein provide novel evidence for effective treatment of ACI by blocking endogenous hepcidin production using the small molecule dorsomorphin derivative LDN-193189 or the protein soluble hemojuvelin-Fc (HJV.Fc) to inhibit bone morphogenetic protein-Smad mediated signaling required for effective hepcidin transcription. Pharmacologic inhibition of hepcidin expression results in mobilization of iron from the RES, stimulation of erythropoiesis and correction of anemia. Thus, hepcidin lowering agents are a promising new class of pharmacologic drugs to effectively combat ACI.

Topics: Anemia; Animals; Antimicrobial Cationic Peptides; Cells, Cultured; Chronic Disease; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Gene Expression; GPI-Linked Proteins; Hemochromatosis Protein; Hepcidins; Immunoglobulin Fc Fragments; Inflammation; Membrane Proteins; Pyrazoles; Pyrimidines; Rats; Rats, Inbred Lew; Remission Induction